Homéoprotéines et plasticité cellulaire / Homeoproteins and cell plasticity

Recherche – Régulation de la sécrétion conventionnelle et non conventionnelle des protéines Page web : https://www.college-de-france.fr/site/en-cirb/joliot-vriz.htm. Le peroxyde d’hydrogène (H2O2) est aujourd’hui considéré comme un élément clé de la signalisation cellulaire. Sa production et sa dégradation sont finement régulées par chaque cellule. Il contribue à la régulation de processus physiologiques notamment en contrôlant le degré d’oxydation des cystéines au sein des protéines. L’éléva..

Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues

<p>Abstract</p> <p>Background</p> <p>Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression.</p> <p>Results</p> <p>We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons.</p> <p>Conclusion</p> <p>We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.</p

Integration of Myeloblastosis Associated Virus proviral sequences occurs in the vicinity of genes encoding signaling proteins and regulators of cell proliferation

AIMS: Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8–10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated. METHODS Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies. RESULTS: The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human. CONCLUSION: The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

AbstractThe active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5 cluster and TyrZ, a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7Å away from the Mn4CaO5 cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I− for Cl− in the cyanobacterium Thermosynechococcus elongatus with either Ca2+ or Sr2+ biosynthetically assembled into the Mn4 cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV–visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl−/I− exchange considerably slowed down the two S3TyrZ•→(S3TyrZ•)′→S0 reactions in which the fast phase, S3TyrZ•→(S3TyrZ•)′, reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on TyrZ• and the slow phase corresponds to the S0 and O2 formations and to a second proton release. The t1/2 for S0 formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the TyrZ• reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca2+ binding site and the Cl− binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl− and Ca2+ binding sites by which proton release may be channelled

Biologie cellulaire des homéoprotéines (2013) | Homéoprotéines et plasticité cellulaire (2014)

    Responsable : Alain Joliot Recherche Mécanismes du transfert intercellulaire des homéoprotéines Au cours de cette année, l’analyse approfondie de lipides spécifiques, les PIP2, dans le transfert intercellulaire des homéoprotéines nous permet de proposer un rôle central de la membrane plasmique dans la répartition des homéoprotéines entre milieu intra- et extracellulaire. En effet, la modulation spécifique de ces lipides au niveau de la membrane plasmique perturbe aussi bien la sécrétion q..

Peptides transducteurs : La face utile d’un nouveau mode de signalisation

En 1988, A.D. Frankel et C.O. Pabo proposent que le facteur de transcription TAT du virus de l’immunodéficience humaine (VIH) est internalisé par les cellules. Parallèlement, des travaux développés dans notre laboratoire démontrent que le domaine de fixation sur l’ADN (l’homéodomaine) d’un facteur de transcription de la famille des homéoprotéines passe directement de l’extérieur vers l’intérieur de la cellule. La séquence responsable de cette internalisation est la troisième hélice de l’homéodomaine, longue de 16 acides aminés. La conservation de cette structure dans tous les facteurs de transcription de la famille des homéoprotéines suggère que l’internalisation est une propriété partagée par la grande majorité de ces protéines. Ces études ont ouvert la voie au concept physiologique de protéine messagère et au développement de peptides de transduction. Ces peptides sont utilisés comme vecteurs pour internaliser dans les cellules, in vitro et in vivo, des cargos hydrophiles de composition et de structure très variées. Cet article fait le point sur les mécanismes impliqués, les utilisations vérifiées ou potentielles de ces peptides et les difficultés rencontrées dans certaines de leurs utilisations, sans oublier les implications physiologiques de ce phénomène.Tranduction peptides that cross the plasma membrane of live cells are commonly used for the in vitro and in vivo targeting of hydrophilic drugs into the cell interior. Although this family of peptides has recently increased and will probably continue to do so, the two mainly used peptides are derived from transcription factors. Indeed, TAT is a 12 amino acid long arginine-rich peptide present in the HIV transcription factor, and penetratin - or its variants - corresponds to 16 amino acids that define the highly conserved third helix of the DNA-binding domain (homeodomain) of homeoprotein transcription factors. In this review, we shall recall the different steps that have led to the discovery of transduction peptides and present the most likely hypotheses concerning the mechanisms involved in their internalization. At the risk of being incomplete or, even, biased, we shall concentrate on penetratins and TAT. The reason is that these peptides have been studied for over ten years leading to the edification of robust knowledge regarding their properties. This attitude will not preclude comparisons with other peptides, if necessary. Our goal is to describe the mode of action of these transduction peptides, their range of activity in term of cell types that accept them and cargoes that they can transport, and, also, some of the limitations that one can encounter in their use. Finally, based on the idea that peptide transduction is the technological face of a physiological property of some transcription factors, we shall discuss the putative physiological function of homeoprotein transduction, and, as a consequence, the possibility to use these factors as therapeutic proteins

Peptides transducteurs

En 1988, A.D. Frankel et C.O. Pabo proposent que le facteur de transcription TAT du virus de l’immunodéficience humaine (VIH) est internalisé par les cellules. Parallèlement, des travaux développés dans notre laboratoire démontrent que le domaine de fixation sur l’ADN (l’homéodomaine) d’un facteur de transcription de la famille des homéoprotéines passe directement de l’extérieur vers l’intérieur de la cellule. La séquence responsable de cette internalisation est la troisième hélice de l’homéodomaine, longue de 16 acides aminés. La conservation de cette structure dans tous les facteurs de transcription de la famille des homéoprotéines suggère que l’internalisation est une propriété partagée par la grande majorité de ces protéines. Ces études ont ouvert la voie au concept physiologique de protéine messagère et au développement de peptides de transduction. Ces peptides sont utilisés comme vecteurs pour internaliser dans les cellules, in vitro et in vivo, des cargos hydrophiles de composition et de structure très variées. Cet article fait le point sur les mécanismes impliqués, les utilisations vérifiées ou potentielles de ces peptides et les difficultés rencontrées dans certaines de leurs utilisations, sans oublier les implications physiologiques de ce phénomène

Homeoprotein transduction in neurodevelopment and physiopathology

International audienceHomeoproteins were originally identified for embryonic cell-autonomous transcription activity, but they also have non-cell-autonomous activity owing to transfer between cells. This Review discusses transfer mechanisms and focuses on some established functions, such as neurodevelopmental regulation of axon guidance, and postnatal critical periods of brain plasticity that affect sensory processing and cognition. Homeoproteins are present across all eukaryotes, and intercellular transfer occurs in plants and animals. Proposed functions have evolutionary relevance, such as morphogenetic activity and sexual exchange during the mating of unicellular eukaryotes, while others have physiopathological relevance, such as regulation of mood and cognition by influencing brain compartmentalization, connectivity, and plasticity. There are more than 250 known homeoproteins with conserved transfer domains, suggesting that this is a common mode of signal transduction but with many undiscovered functions

Mécanismes de sécrétion non-conventionnels des homéoprotéines

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